500 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



centimeter. This emulsion is then shaken for twenty-four nours, 

 filtered or centrifuged, the filtrate preserved with 0.5 per cent, of phenol, 

 and used as antigen. 



Fourth Method. Cultures are grown on a solid medium and washed 

 off with normal saline solution. Saline suspension is then precipitated 

 with an equal quantity of absolute alcohol and centrifugalized. The 

 sediment is dried in vacuo over sulphuric acid, weighed, and ground into 

 a fine powder with sufficient crystals of sodium chlorid to make a 2 per 

 cent suspension of dried material in isotonic saline solution. This 

 stock suspension is not filtered or centrifuged, but is further diluted 

 with saline solution, and constitutes the antigen (method of Besredka, 

 modified by Gay). The actual amounts of dry antigenic substance 

 contained in 1 c.c. of various dilutions are as follows: 



1 c.c. of 1: 40 dilution = 0.5 mg. 



1 c.c. of 1: 80 dilution = 0.25 mg. 



1 c.c. of 1: 160 dilution = 0.125 mg. 



1 c.c. of 1 : 320 dilution = 0.062 mg. 



1 c.c. of 1: 640 dilution = 0.031 mg. 



1 c.c. of 1: 1280 dilution = 1.0155 mg., etc. 



Standardizing Bacterial Antigens. After an antigen has been pre- 

 pared it is standardized by determining the anticomplementary dose 

 i.e., the amount of antigen that just begins to show inhibition of hemol- 

 ysis due to non-specific complement fixation. This dose is easily 

 determined by adding increasing amounts of antigen to a series of test- 

 tubes with a constant dose of complement in each. As a general rule, 

 it is well to add to each tube a constant dose of fresh normal inactivated 

 serum, e. g. } as 0.1 to 0.2 c.c., when the anticomplementary action of 

 serum alone is allowed for. I would emphasize the necessity of doing 

 this in experimental work with rabbit, dog, or any other animal serum. 

 After incubating for one hour, two units of hemolytic amboceptor 

 and 1 c.c. of corpuscle suspension are added to each tube, and the 

 tubes are reincubated for an hour or two and the reading made. In 

 the main test, one-quarter to one-half the anticomplementary unit may 

 be used, as this amount is known to be free from any power of non- 

 specific complement fixation. The former dose is, of course, safer than 

 the latter. 



The standardization may be completed by determining the antigenic 

 dose of the antigen by titrating with a suitable and constant dose of 

 specific immune serum. This titration is conducted by placing in a 

 series of test-tubes increasing doses of antigen with a constant dose of 

 heated immune serum (usually 0.1 c.c.) and a constant dose of comple- 



